Apc conjugated anti il 10

PBMCs were thawed briefly in a 37°C water bath, washed in media (RPMI w/L-glutamine, 10% charcoal inactivated fetal bovine serum, 1% non-essential amino acids, and 1% penicillin/streptomyocin) and counted on a Beckman Coulter Cell Counter (Beckman Coulter, Brea, CA). 10^6 cells were plated per well and incubated at 37°C with 5% CO₂ for 2 hours prior to stimulation. Cells were treated with 2µM ODN2216 (Miltenyi Biotec, Bergisch Gladbach, Germany), 2µM ORN R2336 (Miltenyi Biotec, Bergisch Gladbach, Germany), 2µM ORN R2336 control (Miltenyi Biotec, Bergisch Gladbach, Germany) or media, and returned to incubator. After 2 hours, Golgi Plug (BD biosciences, San Jose, CA) was added to each well, and cells were subsequently incubated for another 4 hours until they were processed for flow cytometry staining as described above. The following antibodies were used in addition to those listed above and were all obtained from Miltenyi Biotec unless otherwise noted (Miltenyi Biotec, Bergisch Gladbach, Germany). PE-conjugated anti-IFNα, PE-conjugated anti-IL-6, PE-conjugated anti-human IgG1, APC-conjugated anti-TNFα, APC-conjugated anti-IL-10, APC-conjugated anti-human IgG1, APC-vio770-conjugated anti-HLA-DR, PerCP Cy5.5-conjugated anti-CD14 (ebioscience inc, Santa Clara, CA).

The cells used for analysis of the CD4⁺ IFNγ ⁺ (Th1), CD4⁺ IL-4⁺ (Th2), CD4⁺ IL-17⁺ (Th17), and IL-17-secreting CD19⁺ B cell (B17) populations were stimulated with PMA and ionomycin for 4 h using GolgiStop (BD Biosciences, Franklin Lake, NJ, USA). To quantify the Th1, Th2, Th17, and B17 cell populations, mouse splenocytes were immunostained using a PerCp5.5-conjugated anti-CD4 antibody (eBioscience, San Diego, CA, USA) for T cells and a PerCP5.5-conjugated anti-CD19 antibody for B cells. The cells were fixed and permeabilized using the Cytofix/Cytoperm Plus kit (BD Biosciences) following the manufacturer’s instructions, and then stained with APC-conjugated anti-IFNγ, PE-conjugated anti-IL-4, APC-conjugated anti-IL-10, and FITC-conjugated anti-IL-17 antibodies (eBioscience), respectively. All samples were analyzed using the Attune NxT Flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA). The flow cytometry data were analyzed using FlowJo™ software (FlowJo, Ashland, OR, USA).